Brassica napus L. var. pabularia (dc.) named KX-1

ABSTRACT

Novel  Brassica napus  L. var. pabularia (DC.), such as  Brassica napus  L. var. pabularia (DC.) designated KX-1 is disclosed. In some embodiments, the invention relates to the seeds of  Brassica napus  L. var. pabularia (DC.) KX-1, to the plants and plant parts of  Brassica napus  L. var. pabularia (DC.) KX-1, and to methods for producing a  Brassica napus  L. var. pabularia (DC.) plant by crossing the  Brassica napus  L. var. pabularia (DC.) KX-1 with itself or another  Brassica napus  L. var. pabularia (DC.) plant. The invention further relates to methods for producing other  Brassica napus  L. var. pabularia (DC.) plants derived from the  Brassica napus  L. var. pabularia (DC.) KX-1.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to, and the benefit of U.S.Provisional Application Ser. No. 62/306,179, filed on Mar. 10, 2017,which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture, to new anddistinctive Brassica napus L. var. pabularia (DC.) cultivar designatedKX-1, and to methods of making and using such plant.

BACKGROUND OF THE INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Brassica napus L. var. pabularia (DC.), also known as Siberian kale isan important and valuable vegetable crop. Thus, a continuing goal ofplant breeders is to develop stable, high yielding Brassica napus L.var. pabularia (DC.) cultivars that are agronomically sound or unique.The reasons for this goal are to maximize the amount of yield producedon the land used as well as to improve the plant agronomic qualities. Toaccomplish this goal, the Brassica napus L. var. pabularia (DC.) breedermust select and develop Brassica napus L. var. pabularia (DC.) plantsthat have the traits that result in superior cultivars.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope.

In various embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

According to the invention, in some embodiments, there is provided anovel Brassica napus L. var. pabularia (DC.) cultivar, designated KX-1.This invention thus relates to the seeds of Brassica napus L. var.pabularia (DC.) cultivar designated KX-1, to the plants or parts thereofof Brassica napus L. var. pabularia (DC.) cultivar designated KX-1, toplants or parts thereof consisting essentially of the phenotypic andmorphological characteristics of Brassica napus L. var. pabularia (DC.)cultivar designated KX-1, and/or having all the physiological andmorphological characteristics of Brassica napus L. var. pabularia (DC.)cultivar designated KX-1 and/or having one or more or all of thecharacteristics of Brassica napus L. var. pabularia (DC.) cultivardesignated KX-1 listed in Table 1 including, but not limited to, asdetermined at the 5% significance level when grown in the sameenvironmental conditions, and/or having one or more of the physiologicaland morphological characteristics of Brassica napus L. var. pabularia(DC.) cultivar designated KX-1 listed in Table 1 including, but notlimited to, as determined at the 5% significance level when grown in thesame environmental conditions, and/or having all the physiological andmorphological characteristics of Brassica napus L. var. pabularia (DC.)cultivar designated KX-1 listed in Table 1 including but not limited toas determined at the 5% significance level when grown in the sameenvironmental conditions and/or having all the physiological andmorphological characteristics of Brassica napus L. var. pabularia (DC.)cultivar designated KX-1 listed in Table 1 when grown in the sameenvironmental conditions. The invention also relates to variants,mutants and trivial modifications of the seed or plant of Brassica napusL. var. pabularia (DC.) cultivar designated KX-1.

Plant parts of the Brassica napus L. var. pabularia (DC.) cultivar ofthe present invention are also provided, such as a leaf, flower, cell,pollen or ovule obtained from the plant cultivar. The present inventionprovides leaves of the Brassica napus L. var. pabularia (DC.) cultivarof the present invention. Such leaves could be used as fresh productsfor consumption or in processes resulting in processed products such asfood products comprising one or more harvested part of the Brassicanapus L. var. pabularia (DC.) plant KX-1, for example harvested leaves.The harvested part or food product can be or can comprise the Brassicanapus L. var. pabularia (DC.) leaves of the Brassica napus L. var.pabularia (DC.) plant KX-1 or a salad mixture comprising leaves of theBrassica napus L. var. pabularia (DC.) plant KX-1. The food productsmight have undergone one or more processing steps such as, but notlimited to cutting, washing, mixing, etc. All such products are part ofthe present invention.

The plants and seeds of the present invention include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act of the United States of America, i.e., avariety that is predominantly derived from Brassica napus L. var.pabularia (DC.) cultivar designated KX-1 or from a variety that i) ispredominantly derived from Brassica napus L. var. pabularia (DC.)cultivar designated KX-1, while retaining the expression of theessential characteristics that result from the genotype or combinationof genotypes of Brassica napus L. var. pabularia (DC.) cultivardesignated KX-1; ii) is clearly distinguishable from Brassica napus L.var. pabularia (DC.) cultivar designated KX-1; and iii) except fordifferences that result from the act of derivation, conforms to theinitial variety in the expression of the essential characteristics thatresult from the genotype or combination of genotypes of the initialvariety or cultivar.

In another aspect, the present invention provides regenerable cells. Insome embodiments, the regenerable cells are for use in tissue culture ofBrassica napus L. var. pabularia (DC.) cultivar designated KX-1. In someembodiments, the tissue culture is capable of regenerating plantsconsisting essentially of the phenotypic and morphologicalcharacteristics of Brassica napus L. var. pabularia (DC.) cultivardesignated KX-1, and/or having all the phenotypic and morphologicalcharacteristics of Brassica napus L. var. pabularia (DC.) cultivardesignated KX-1, and/or having the physiological and morphologicalcharacteristics of Brassica napus L. var. pabularia (DC.) cultivardesignated KX-1, and/or having the characteristics of Brassica napus L.var. pabularia (DC.) cultivar designated KX-1. In one embodiment, theregenerated plants have the characteristics of Brassica napus L. var.pabularia (DC.) cultivar designated KX-1 listed in Table 1 including butnot limited to as determined at the 5% significance level when grown inthe same environmental conditions. In some embodiments, the plant partsand cells used to produce such tissue cultures can be embryos,meristematic cells, seeds, callus, pollen, leaves, anthers, pistils,roots, root tips, stems, petioles, cotyledons, hypocotyls, ovaries, seedcoat, fruits, endosperm, flowers, axillary buds or the like. Protoplastsproduced from such tissue culture are also included in the presentinvention. The Brassica napus L. var. pabularia (DC.) shoots, roots andwhole plants regenerated from the tissue culture, as well as the leavesproduced by said regenerated plants are also part of the invention. Insome embodiments, the whole plants regenerated from the tissue culturehave one, more than one, or all of the physiological and morphologicalcharacteristics of Brassica napus L. var. pabularia (DC.) cultivardesignated KX-1 listed in Table 1 including but not limited to whengrown in the same environmental conditions.

The invention also discloses methods for vegetatively propagating aplant of the present invention. In some embodiments, the methodscomprise collecting a part of a Brassica napus L. var. pabularia (DC.)cultivar designated KX-1 and regenerating a plant from said part. Insome embodiments, the part can be for example a leaf cutting that isrooted into an appropriate medium according to techniques known by theone skilled in the art. Plants and plant parts thereof such as leavesproduced by such methods are also included in the present invention. Inanother aspect, the plants thereof produced by such methods consistessentially of the phenotypic and morphological characteristics ofBrassica napus L. var. pabularia (DC.) cultivar designated KX-1, and/orhaving all the phenotypic and morphological characteristics of Brassicanapus L. var. pabularia (DC.) cultivar designated KX-1, and/or havingthe physiological and morphological characteristics of Brassica napus L.var. pabularia (DC.) cultivar designated KX-1, and/or having thecharacteristics of Brassica napus L. var. pabularia (DC.) cultivardesignated KX-1. In some embodiments, plants produced by such methodsconsist of one, more than one, or all phenotypic and morphologicalcharacteristics of Brassica napus L. var. pabularia (DC.) cultivardesignated KX-1 listed in Table 1 including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

Further included in the invention are methods for producing leaves fromthe Brassica napus L. var. pabularia (DC.) cultivar designated KX-1. Insome embodiments, the methods comprise growing a Brassica napus L. var.pabularia (DC.) cultivar designated KX-1 to produce a Brassica napus L.var. pabularia (DC.) leaf. In some embodiments, the methods furthercomprise harvesting the Brassica napus L. var. pabularia (DC.) leaf. Insome embodiments, the methods further comprise harvesting the Brassicanapus L. var. pabularia (DC.) leaf at an early stage of production,leading to “baby leaf” harvest. Such Brassica napus L. var. pabularia(DC.) leaves, at any stage of production, are part of the presentinvention.

Also included in this invention are methods for producing a Brassicanapus L. var. pabularia (DC.) plant. In some embodiments, the Brassicanapus L. var. pabularia (DC.) plant is produced by crossing the Brassicanapus L. var. pabularia (DC.) cultivar designated KX-1 with itself oranother plant. In some embodiments, the other plant can be a Brassicanapus L. var. pabularia (DC.) plant. In some embodiments, the otherplant can be a Brassica napus L. var. pabularia (DC.) hybrid or line.When crossed with itself, i.e. when KX-1 is crossed with anotherBrassica napus L. var. pabularia (DC.) cultivar KX-1 or self-pollinated,Brassica napus L. var. pabularia (DC.) cultivar KX-1 will be conserved(e.g. as an inbred). When crossed with another, different Brassica napusL. var. pabularia (DC.) plant, an F1 hybrid seed is produced if thedifferent Brassica napus L. var. pabularia (DC.) plant is an inbred anda “three-way cross” seed is produced if the different Brassica napus L.var. pabularia (DC.) plant is a hybrid. Such F1 hybrid seed andthree-way hybrid seeds and plants produced by growing said F1 andthree-way hybrid seeds are included in the present invention. Methodsfor producing an F1 and three-way hybrid Brassica napus L. var.pabularia (DC.) seed comprising crossing Brassica napus L. var.pabularia (DC.) cultivar KX-1 plant with a different Brassica napus L.var. pabularia (DC.) line or hybrid and harvesting the resultant hybridBrassica napus L. var. pabularia (DC.) seed are also part of theinvention. The hybrid Brassica napus L. var. pabularia (DC.) seedsproduced by the methods comprising crossing Brassica napus L. var.pabularia (DC.) cultivar KX-1 plant with a different Brassica napus L.var. pabularia (DC.) plant and harvesting the resultant hybrid Brassicanapus L. var. pabularia (DC.) seed are included in the invention, as areincluded the hybrid Brassica napus L. var. pabularia (DC.) plants orparts thereof and seeds produced by said grown hybrid Brassica napus L.var. pabularia (DC.) plants.

Also included in this invention are methods for producing a hybridBrassica plant. In some embodiments, the hybrid Brassica plant isproduced by crossing the Brassica napus L. var. pabularia (DC.) cultivardesignated KX-1 with another plant. In some embodiments, the other plantcan be a variety of the Brassica plant species selected from the groupconsisting of Brassica napus (e.g., rutabaga, Siberian kale), Brassicarapa (e.g., Chinese cabbage, pai-tsai, mizuna, Chinese mustard, broccoliraab, and turnip), Brassica oleracea (e.g., cabbage, broccoli,cauliflower, Brussels sprouts, kohlrabi, collards, kale), Raphanussativus (e.g., radish), Brassica nigra (e.g., black mustard), Brassicacarinata (e.g., Ethiopian mustard), Brassica juncea (e.g., brown orIndian mustard), and Armoracea rusticana (e.g., horseradish). Whencrossed with another, different Brassica plant, an F1 hybrid seed isproduced if the different Brassica plant is an inbred and a “three-waycross” seed is produced if the different Brassica plant is a hybrid.Such F1 hybrid seed and three-way hybrid seeds and plants produced bygrowing said F1 and three-way hybrid seeds are included in the presentinvention. Methods for producing an F1 and three-way hybrid Brassicaseed comprising crossing Brassica napus L. var. pabularia (DC.) cultivarKX-1 plant with a different Brassica line or hybrid and harvesting theresultant hybrid Brassica seed are also part of the invention. Thehybrid Brassica seeds produced by the methods comprising crossingBrassica napus L. var. pabularia (DC.) cultivar KX-1 plant with adifferent Brassica plant and harvesting the resultant hybrid Brassicaseed are included in the invention, as are included the hybrid plants orparts thereof and seeds produced by said grown hybrid Brassica plants.

Further included in the invention are methods for producing a Brassicanapus L. var. pabularia (DC.) seed and plants made thereof. In someembodiments, said methods comprise self-pollinating the Brassica napusL. var. pabularia (DC.) cultivar KX-1 and harvesting the resultantseeds. Brassica napus L. var. pabularia (DC.) seeds produced by suchmethod are also part of the invention.

In another embodiment, this invention also relates to methods forproducing other Brassica napus L. var. pabularia (DC.) plants derivedfrom Brassica napus L. var. pabularia (DC.) cultivar KX-1 and to theBrassica napus L. var. pabularia (DC.) plants derived by the use ofthose methods.

In some embodiments, such methods for producing a Brassica napus L. var.pabularia (DC.) plant derived from the Brassica napus L. var. pabularia(DC.) cultivar KX-1 comprise (a) self-pollinating the Brassica napus L.var. pabularia (DC.) cultivar KX-1 plant at least once to produce aprogeny plant derived from Brassica napus L. var. pabularia (DC.)cultivar KX-1. In some embodiments, the methods further comprise (b)crossing the progeny plant derived from Brassica napus L. var. pabularia(DC.) cultivar KX-1 with itself or a second Brassica napus L. var.pabularia (DC.) plant to produce a seed of a progeny plant of asubsequent generation. In some embodiments, the methods further comprise(c) growing the progeny plant of the subsequent generation. In someembodiments, the method further comprises (d) crossing the progeny plantof the subsequent generation with itself or a second Brassica napus L.var. pabularia (DC.) plant to produce a Brassica napus L. var. pabularia(DC.) plant further derived from the Brassica napus L. var. pabularia(DC.) cultivar KX-1. In further embodiments, step (b), step (c) and/orstep (d) are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, or moregenerations to produce a Brassica napus L. var. pabularia (DC.) plantderived from the Brassica napus L. var. pabularia (DC.) cultivar KX-1.In some embodiments, within each crossing cycle, the second plant is thesame plant as the second plant in the last crossing cycle. In someembodiment, within each crossing cycle, the second plant is differentfrom the second plant of the last crossing cycle.

Another method for producing a Brassica napus L. var. pabularia (DC.)plant derived from the variety KX-1, comprises the steps of: (a)crossing the KX-1 plant with a second Brassica napus L. var. pabularia(DC.) plant to produce a progeny plant derived from Brassica napus L.var. pabularia (DC.) cultivar KX-1. In some embodiments, the methodfurther comprises (b) crossing the progeny plant derived from Brassicanapus L. var. pabularia (DC.) cultivar KX-1 with itself or a secondBrassica napus L. var. pabularia (DC.) plant to produce a seed of aprogeny plant of a subsequent generation. In some embodiments, themethod further comprises (c) growing the progeny plant of the subsequentgeneration from the seed; In some embodiments, the method furthercomprises (d) crossing the progeny plant of the subsequent generationwith itself or a different Brassica napus L. var. pabularia (DC.) plantto produce a Brassica napus L. var. pabularia (DC.) plant derived fromKX-1. In a further embodiment, step (b), step (c) and/or step (d) arerepeated for at least 1, 2, 3, 4, 5, 6, 7, 8, or more generations toproduce a Brassica napus L. var. pabularia (DC.) plant derived fromKX-1. In some embodiments, within each crossing cycle, the second plantis the same plant as the second plant in the last crossing cycle. Insome embodiments, within each crossing cycle, the second plant isdifferent from the second plant in the last crossing cycle.

In another aspect, the present invention provides methods of introducingor modifying one or more desired trait(s) into the Brassica napus L.var. pabularia (DC.) cultivar KX-1 and plants or seeds obtained fromsuch methods. The desired trait(s) may be, but not exclusively, a singlegene. In some embodiments, the gene is a dominant allele. In someembodiments, the gene is a partially dominant allele. In someembodiments, the gene is a recessive allele. In some embodiments, thegene or genes will confer such traits as male sterility, herbicideresistance, insect resistance, resistance for bacterial, fungal,mycoplasma or viral disease, improved shelf life, water-stresstolerance, delayed senescence or controlled ripening, enhanced plantquality such as improved drought or salt tolerance, enhanced plantvigor, improved or changed colors or improved fresh cut application. Forthe present invention and the skilled artisan, disease is understood toinclude, but not limited to fungal diseases, viral diseases, bacterialdiseases, mycoplasma diseases, or other plant pathogenic diseases and adisease resistant plant will encompass a plant resistant to fungal,viral, bacterial, mycoplasma, and other plant pathogens. The gene orgenes may be naturally occurring Brassica napus L. var. pabularia (DC.)gene(s), mutant(s) or genes modified through New Breeding Techniques. Insome embodiments, the method for introducing the desired trait(s) is abackcrossing process making use of a series of backcrosses to Brassicanapus L. var. pabularia (DC.) cultivar KX-1 during which the desiredtrait(s) is maintained by selection. The single gene conversion plantsthat can be obtained by the method are included in the presentinvention. In some embodiments, the desired trait is a transgene.

When dealing with a gene that has been modified, for example through NewBreeding Techniques, the trait (genetic modification) could be directlymodified into the newly developed line/cultivar such as Brassica napusL. var. pabularia (DC.) cultivarKX-1. Alternatively, if the trait is notmodified into each newly developed line/cultivar such as lettucecultivar KX-1, another typical method used by breeders of ordinary skillin the art to incorporate the modified gene is to take a plant alreadycarrying the gene and to use such plant as a donor line to transfer thegene into the newly developed line.

The same would apply for a naturally occurring trait or one arising fromspontaneous or induced mutations.

In some embodiments, the backcross breeding process of Brassica napus L.var. pabularia (DC.) cultivar KX-1 comprises (a) crossing Brassica napusL. var. pabularia (DC.) cultivar KX-1 with plants that comprise thedesired trait(s) to produce F1 progeny plants. In some embodiments, theprocess further comprises (b) selecting the F 1 progeny plants that havethe desired trait(s). In some embodiments, the process further comprises(c) crossing the selected F1 progeny plants with the Brassica napus L.var. pabularia (DC.) cultivar KX-1 plants to produce backcross progenyplants. In some embodiments, the process further comprises (d) selectingfor backcross progeny plants that have the desired trait(s) andphysiological and morphological characteristics of the Brassica napus L.var. pabularia (DC.) cultivar KX-1 to produce selected backcross progenyplants. In some embodiments, the process further comprises (e) repeatingsteps (c) and (d) one, two, three, four, five six, seven, eight, nine ormore times in succession to produce selected, second, third, fourth,fifth, sixth, seventh, eighth, ninth or higher backcross progeny plantsthat have the desired trait(s) and consist essentially of the phenotypicand morphological characteristics of the Brassica napus L. var.pabularia (DC.) cultivar KX-1, and/or have all the phenotypic andmorphological characteristics of the Brassica napus L. var. pabularia(DC.) cultivar KX-1, and/or have the desired trait(s) and thephysiological and morphological characteristics of the Brassica napus L.var. pabularia (DC.) cultivar KX-1 as determined in Table 1, includingbut not limited to when grown in the same environmental conditions orincluding but not limited to at a 5% significance level when grown inthe same environmental conditions. In some embodiments, the backcrossbreeding process of Brassica napus L. var. pabularia (DC.) cultivar ofKX-1 comprises the following steps: (a) crossing Brassica napus L. var.pabularia (DC.) cultivar KX-1 with plants of another line that comprisethe desired trait(s) to produce F 1 progeny plants, (b) selecting the F1 progeny plants that have the desired trait(s); (c) crossing theselected F1 progeny plants with the Brassica napus L. var. pabularia(DC.) cultivar KX-1 plants to produce backcross progeny plants; (d)selecting for backcross progeny plants that have the desired trait(s)and physiological and morphological characteristics of the Brassicanapus L. var. pabularia (DC.) cultivar KX-1 to produce selectedbackcross progeny plants; and (e) repeating steps (c) and (d) one, two,three, four, five six, seven, eight, nine or more times in succession toproduce selected, second, third, fourth, fifth, sixth, seventh, eighth,ninth or higher backcross progeny plants that consist essentially of thephenotypic and morphological characteristics of the Brassica napus L.var. pabularia (DC.) cultivar KX-1, and/or have all the phenotypic andmorphological characteristics of the Brassica napus L. var. pabularia(DC.) cultivar KX-1, and/or have the desired trait(s) and thephysiological and morphological characteristics of the Brassica napus L.var. pabularia (DC.) cultivar KX-1 as determined in Table 1, includingbut not limited to at a 5% significance level when grown in the sameenvironmental conditions. The Brassica napus L. var. pabularia (DC.)plants or seed produced by the methods are also part of the invention.Backcrossing breeding methods, well known to one skilled in the art ofplant breeding will be further developed in subsequent parts of thespecification.

In an embodiment of this invention is a method of making a backcrossconversion of Brassica napus L. var. pabularia (DC.) cultivar KX-1. Insome embodiments, the method comprises crossing Brassica napus L. var.pabularia (DC.) cultivar KX-1 with a donor plant comprising a mutantgene(s), a naturally occurring gene(s) or a gene(s) and/or sequencesmodified through the use of New Breeding Techniques conferring one ormore desired trait to produce F1 progeny plant. In some embodiment, themethod further comprises selecting the F1 progeny plant comprising thenaturally occurring gene(s), mutant gene(s) or modified gene(s) and/orsequences conferring the one or more desired trait. In some embodiments,the method further comprises backcrossing the selected progeny plant tothe Brassica napus L. var. pabularia (DC.) cultivar KX-1. This methodmay further comprise the step of obtaining a molecular marker profile ofthe Brassica napus L. var. pabularia (DC.) cultivar KX-1 and using themolecular marker profile to select for the progeny plant with thedesired trait and the molecular marker profile of the Brassica napus L.var. pabularia (DC.) cultivar KX-1. The plants or parts thereof producedby such methods are also part of the present invention.

In some embodiments of the invention, the number of loci that may bebackcrossed into the Brassica napus L. var. pabularia (DC.) cultivarKX-1 is at least 1, 2, 3, 4, 5 or more. A single locus may contain oneor several genes. A single locus conversion also allows for making oneor more site specific changes to the plant genome, such as, withoutlimitation, one or more nucleotide change, deletion, insertions, etc. Insome embodiments, the single locus conversion is performed by genomeediting, a.k.a. genome editing with engineered nucleases (GEEN). In someembodiments, the genome editing comprises using one or more engineerednucleases. In some embodiments, the engineered nucleases include, butare not limited to Zinc finger nucleases (ZFNs), TranscriptionActivator-Like Effector Nucleases (TALENs), the CRISPR/Cas system,engineered meganuclease re-engineered homing endonucleases, andendonucleases for DNA guided genome editing (Gao et al., NatureBiotechnology (2016), doi: 10.1038/nbt.3547). In some embodiments, thesingle locus conversion changes one or several nucleotides of the plantgenome. Such genome editing techniques are some of the techniques nowknown by a person skilled in the art and herein are collectivelyreferred to as ‘New Breeding Techniques’.

The invention further provides methods for developing Brassica napus L.var. pabularia (DC.) plants in a Brassica napus L. var. pabularia (DC.)plant breeding program using plant breeding techniques including but notlimited to, recurrent selection, backcrossing, pedigree breeding,genomic selection, molecular marker (Isozyme Electrophoresis,Restriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), and Simple Sequence Repeats (SSRs) which are also referred toas Microsatellites, Single Nucleotide Polymorphisms (SNPs), etc.)enhanced selection, genetic marker enhanced selection andtransformation. Seeds, Brassica napus L. var. pabularia (DC.) plants,and parts thereof produced by such breeding methods are also part of theinvention.

The invention also relates to variants, mutants and trivialmodifications of the seed or plant of the Brassica napus L. var.pabularia (DC.) cultivar KX-1. Variants, mutants and trivialmodifications of the seed or plant of Brassica napus L. var. pabularia(DC.) cultivar KX-1 can be generated by methods available to one skilledin the art, including but not limited to, mutagenesis (e.g., chemicalmutagenesis, radiation mutagenesis, transposon mutagenesis, insertionalmutagenesis, signature tagged mutagenesis, site-directed mutagenesis,and natural mutagenesis), knock-outs/knock-ins, antisense RNAinterference and other techniques such as the New Breeding Techniques.For more information of mutagenesis in plants, such as agents,protocols, see Acquaah et al. (Principles of plant genetics andbreeding, Wiley-Blackwell, 2007, ISBN 1405136464, 9781405136464, whichis herein incorporated by reference in its entity).

The invention also relates to a mutagenized population of the Brassicanapus L. var. pabularia (DC.) cultivar KX-1 and methods of using suchpopulations. In some embodiments, the mutagenized population can be usedin screening for new Brassica napus L. var. pabularia (DC.) plants whichcomprises one or more or all of the morphological and physiologicalcharacteristics of Brassica napus L. var. pabularia (DC.) cultivar KX-1.In some embodiments, the new Brassica napus L. var. pabularia (DC.)plants obtained from the screening process comprise all of themorphological and physiological characteristics of the Brassica napus L.var. pabularia (DC.) cultivar KX-1, and one or more additional ordifferent morphological and physiological characteristics that Brassicanapus L. var. pabularia (DC.) cultivar KX-1 does not have.

This invention also is directed to methods for producing a Brassicanapus L. var. pabularia (DC.) plant by crossing a first parent Brassicanapus L. var. pabularia (DC.) plant with a second parent Brassica napusL. var. pabularia (DC.) plant wherein either the first or second parentBrassica napus L. var. pabularia (DC.) plant is a Brassica napus L. var.pabularia (DC.) cultivar KX-1. Further, both first and second parentBrassica napus L. var. pabularia (DC.) plants can come from the Brassicanapus L. var. pabularia (DC.) cultivar KX-1. Further, the Brassica napusL. var. pabularia (DC.) cultivar KX-1 can be self-pollinated i.e. thepollen of a Brassica napus L. var. pabularia (DC.) cultivar KX-1 canpollinate the ovule of the same Brassica napus L. var. pabularia (DC.)cultivar KX-1, respectively. When crossed with another Brassica napus L.var. pabularia (DC.) plant, a hybrid seed is produced. Such methods ofhybridization and self-pollination are well known to those skilled inthe art of breeding.

A Brassica napus L. var. pabularia (DC.) cultivar such as Brassica napusL. var. pabularia (DC.) cultivar KX-1 has been produced through severalcycles of self-pollination and is therefore to be considered as ahomozygous plant or line. An inbred line can also be produced throughthe dihaploid system which involves doubling the chromosomes from ahaploid plant or embryo thus resulting in an inbred line that isgenetically stable (homozygous) and can be reproduced without alteringthe inbred line: Haploid plants could be obtained from haploid embryosthat might be produced from microspores, pollen, anther cultures orovary cultures or spontaneous haploidy. The haploid embryos may then bedoubled by chemical treatments such as by colchicine or be doubledautonomously. The haploid embryos may also be grown into haploid plantsand treated to induce the chromosome doubling. In either case, fertilehomozygous plants are obtained. A hybrid variety is classically createdthrough the fertilization of an ovule from an inbred parental line bythe pollen of another, different inbred parental line. Due to thehomozygous state of the inbred line, the produced gametes carry a copyof each parental chromosome. As both the ovule and the pollen bring acopy of the arrangement and organization of the genes present in theparental lines, the genome of each parental line is present in theresulting F1 hybrid, theoretically in the arrangement and organizationcreated by the plant breeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting F1 hybrid cross shall be stable. The F1 hybrid is then acombination of phenotypic characteristics issued from two arrangementand organization of genes, both created by a person skilled in the artthrough the breeding process.

Still further, this invention also is directed to methods for producinga Brassica napus L. var. pabularia (DC.) plant derived from Brassicanapus L. var. pabularia (DC.) plant cultivar KX-1 by crossing Brassicanapus L. var. pabularia (DC.) cultivar KX-1 with a second Brassica napusL. var. pabularia (DC.) plant. In some embodiments, the method furthercomprises obtaining a progeny seed from the cross. In some embodiment,the method further comprises growing the progeny seed, and possiblyrepeating the crossing and growing steps with the Brassica napus L. var.pabularia (DC.) cultivar KX-1 -derived plant from 0 to 7, or more times.Thus, any such methods using the Brassica napus L. var. pabularia (DC.)cultivar KX-1 are part of this invention: selfing, backcrosses, hybridproduction, crosses to populations, and the like. All plants producedusing Brassica napus L. var. pabularia (DC.) cultivar KX-1 as a parentare within the scope of this invention, including plants derived fromBrassica napus L. var. pabularia (DC.) cultivar KX-1. In someembodiment, such plants have one, more than one, or all phenotypic andmorphological characteristics of Brassica napus L. var. pabularia (DC.)cultivar KX-1 listed in Table 1 including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

Such plants might exhibit additional and desired characteristics ortraits such as high seed yield, high seed germination, seedling vigor,early maturity, high yield, disease tolerance or resistance, andadaptability for soil and climate conditions. Consumer-driven traits,such as a preference for a given leaf size, shape, color, texture,taste, are other traits that may be incorporated into new Brassica napusL. var. pabularia (DC.) plants developed by this invention.

A Brassica napus L. var. pabularia (DC.) plant can also be propagatedvegetatively. A part of the plant, for example a shoot tissue, iscollected, and a new plant is obtained from the part. Such parttypically comprises an apical meristem of the plant. The collected partis transferred to a medium allowing development of a plantlet, includingfor example rooting or development of shoots. This is achieved usingmethods well-known in the art. Accordingly, in one embodiment, a methodof vegetatively propagating a plant of the present invention comprisescollecting a part of a plant according to the present invention, e.g. ashoot tissue, and obtaining a plantlet from said part. In oneembodiment, a method of vegetatively propagating a plant of the presentinvention comprises: a) collecting tissue of a plant of the presentinvention; b) rooting said proliferated shoots to obtain rootedplantlets. In one embodiment, a method of vegetatively propagating aplant of the present invention comprises: a) collecting tissue of aplant of the present invention; b) cultivating said tissue to obtainproliferated shoots; c) rooting said proliferated shoots to obtainrooted plantlets. In one embodiment, such method further comprisesgrowing a plant from said plantlets. In one embodiment, leaves areharvested from said plant. In one embodiment, the leaves are processedinto products prepared cut leaves.

In some embodiments, the present invention teaches a seed of Brassicanapus L. var. pabularia (DC.) cultivar KX-1, wherein a representativesample of seed of said Brassica napus L. var. pabularia (DC.) cultivaris deposited under NCIMB No 42703 .

In some embodiments, the present invention teaches a Brassica napus L.var. pabularia (DC.) plant, or a part thereof, produced by growing thedeposited KX-1 seed.

In some embodiments, the present invention teaches Brassica napus L.var. pabularia (DC.) plant parts, wherein the Brassica napus L. var.pabularia (DC.) part is selected from the group consisting of: a leaf, aflower, an ovule, pollen, and a cell.

In some embodiments, the present invention teaches a Brassica napus L.var. pabularia (DC.) plant, or a part thereof, having all of thecharacteristics of Brassica napus L. var. pabularia (DC.) cultivar KX-1as listed in Table 1 of this application including but not limited towhen grown in the same environmental conditions.

In some embodiments, the present invention teaches a Brassica napus L.var. pabularia (DC.) plant, or a part thereof, having all of thephysiological and morphological characteristics of Brassica napus L.var. pabularia (DC.) cultivar KX-1, wherein a representative sample ofseed of said Brassica napus L. var. pabularia (DC.) plant was depositedunder NCIMB No 42703.

In some embodiments, the present invention teaches a compositioncomprising regenerable cells produced from the plant or plant part grownfrom the deposited Brassica napus L. var. pabularia (DC.) cultivar KX-1seed, or other plant part or plant cell. In some embodiments, thecomposition comprises a growth media. In some embodiments, the growthmedia is solid or a synthetic cultivation medium. In some embodiments,the composition is tissue culture of regenerable cells produced from theplant or plant part grown from the deposited Brassica napus L. var.pabularia (DC.) cultivar KX-1 seed, wherein cells of the tissue cultureare produced from a plant part selected from the group consisting ofprotoplasts, embryos, meristematic cells, callus, pollen, ovules,flowers, seeds, leaves, roots, root tips, anthers, stems, petioles,head, axillary buds, cotyledons and hypocotyls. In some embodiments, theplant part includes protoplasts produced from a plant grown from thedeposited Brassica napus L. var. pabularia (DC.) cultivar KX-1 seed.

In some embodiments, the present invention teaches a Brassica napus L.var. pabularia (DC.) plant regenerated from the tissue culture from aplant grown from the deposited Brassica napus L. var. pabularia (DC.)cultivar KX-1 seed, said plant having the characteristics of Brassicanapus L. var. pabularia (DC.) cultivar KX-1, wherein a representativesample of seed of said Brassica napus L. var. pabularia (DC.) cultivarKX-1 is deposited under NCIMB No 42703.

In some embodiments, the present invention teaches a Brassica napus L.var. pabularia (DC.) leaf produced from plants grown from the depositedBrassica napus L. var. pabularia (DC.) cultivar KX-1 seed.

In some embodiments, the methods of producing said Brassica napus L.var. pabularia (DC.) leaf comprise a) growing the Brassica napus L. var.pabularia (DC.) plant from deposited Brassica napus L. var. pabularia(DC.) cultivar KX-1 seed to produce a Brassica napus L. var. pabularia(DC.) leaf, and b) harvesting said Brassica napus L. var. pabularia(DC.) leaf. In some embodiments, the present invention also teaches aBrassica napus L. var. pabularia (DC.) leaf produced by the method ofproducing Brassica napus L. var. pabularia (DC.) leaf as describedabove.

In some embodiments, the present invention teaches methods for producinga Brassica napus L. var. pabularia (DC.) seed comprising crossing afirst parent Brassica napus L. var. pabularia (DC.) plant with a secondparent Brassica napus L. var. pabularia (DC.) plant and harvesting theresultant Brassica napus L. var. pabularia (DC.) seed, wherein saidfirst parent Brassica napus L. var. pabularia (DC.) plant and/or secondparent Brassica napus L. var. pabularia (DC.) plant is the Brassicanapus L. var. pabularia (DC.) plant produced from the deposited Brassicanapus L. var. pabularia (DC.) cultivar KX-1 seed, or a Brassica napus L.var. pabularia (DC.) plant having all of the characteristics of Brassicanapus L. var. pabularia (DC.) cultivar KX-1 as listed in Table 1 of thisapplication, including but not limited to when grown in the sameenvironmental conditions

In some embodiments, the present invention teaches methods for producinga Brassica napus L. var. pabularia (DC.) seed comprisingself-pollinating the Brassica napus L. var. pabularia (DC.) plant grownfrom the deposited Brassica napus L. var. pabularia (DC.) cultivar KX-1seed and harvesting the resultant Brassica napus L. var. pabularia (DC.)seed.

In some embodiments, the present invention teaches the seed produced byany of the above described methods.

In some embodiments, the present invention teaches methods ofvegetatively propagating the Brassica napus L. var. pabularia (DC.)plant grown from the deposited Brassica napus L. var. pabularia (DC.)cultivar KX-1 seed, said method comprising a) collecting part of a plantgrown from the deposited Brassica napus L. var. pabularia (DC.) cultivarKX-1 seed and b) regenerating a plant from said part.

In some embodiments, the method further comprises harvesting a leaf fromsaid vegetatively propagated plant.

In some embodiments, the present invention teaches the plant, the leavesthereof of plants vegetatively propagated from plant parts of plantsgrown from the deposited Brassica napus L. var. pabularia (DC.) cultivarKX-1 seed.

In some embodiments, the present invention teaches methods of producinga Brassica napus L. var. pabularia (DC.) plant derived from the Brassicanapus L. var. pabularia (DC.) cultivar PYTHON. In some embodiments, themethods comprises (a) self-pollinating the plant grown from thedeposited Brassica napus L. var. pabularia (DC.) cultivar KX-1 seed atleast once to produce a progeny plant derived from Brassica napus L.var. pabularia (DC.) cultivar KX-1. In some embodiment, the methodfurther comprise (b) crossing the progeny plant derived from Brassicanapus L. var. pabularia (DC.) cultivar KX-1 with itself or a secondBrassica napus L. var. pabularia (DC.) plant to produce a seed of aprogeny plant of a subsequent generation; and (c) growing the progenyplant of the subsequent generation from the seed, and crossing theprogeny plant of the subsequent generation with itself or a secondBrassica napus L. var. pabularia (DC.) plant to produce a Brassica napusL. var. pabularia (DC.) plant derived from the Brassica napus L. var.pabularia (DC.) cultivar KX-1. In some embodiments said method furthercomprises the step of: (d) repeating steps b) and/or c) for at least 1,2, 3, 4, 5, 6, 7, or more generation to produce a Brassica napus L. var.pabularia (DC.) plant derived from the Brassica napus L. var. pabularia(DC.) cultivar KX-1.

In some embodiments, the present invention teaches methods of producinga Brassica napus L. var. pabularia (DC.) plant derived from the Brassicanapus L. var. pabularia (DC.) cultivar KX-1, the methods comprising (a)crossing the plant grown from the deposited Brassica napus L. var.pabularia (DC.) cultivar KX-1 seed with a second Brassica napus L. var.pabularia (DC.) plant to produce a progeny plant derived from Brassicanapus L. var. pabularia (DC.) cultivar KX-1. In some embodiments, themethods further comprise (b) crossing the progeny plant derived fromBrassica napus L. var. pabularia (DC.) cultivar KX-1 with itself or asecond Brassica napus L. var. pabularia (DC.) plant to produce a seed ofa progeny plant of a subsequent generation; and (c) growing the progenyplant of the subsequent generation from the seed and crossing theprogeny plant of the subsequent generation with itself or a secondBrassica napus L. var. pabularia (DC.) plant to produce a Brassica napusL. var. pabularia (DC.) plant derived from the Brassica napus L. var.pabularia (DC.) cultivar KX-1. In some embodiments said method furthercomprises the step of: (d) repeating steps (b) and/or (c) for at least1, 2, 3, 4, 5, 6, 7 or more generation to produce a Brassica napus L.var. pabularia (DC.) plant derived from the Brassica napus L. var.pabularia (DC.) cultivar KX-1.

In some embodiments, the present invention teaches plants grown from thedeposited Brassica napus L. var. pabularia (DC.) cultivar KX-1 seedwherein said plants comprise a single locus conversion. As used herein,the term “a” or “an” refers to one or more of that entity; for example,“a single locus conversion” refers to one or more single locusconversions or at least one single locus conversion. As such, the terms“a” (or “an”), “one or more” and “at least one” are used interchangeablyherein. In addition, reference to “an element” by the indefinite article“a” or “an” does not exclude the possibility that more than one of theelements are present, unless the context clearly requires that there isone and only one of the elements. In some embodiments said single locusconversion confers said plant with a trait selected from the groupconsisting of male sterility, male fertility, herbicide resistance,insect resistance, disease resistance, water stress tolerance, heattolerance, delayed senescence, improved ripening control, long shelflife, and improved salt tolerance when compared to a suitable checkplant. In some embodiments, the check plant is a Brassica napus L. var.pabularia (DC.) cultivar KX-1 plant not having said single locusconversion. In some embodiments, the single locus conversion is anartificially mutated gene or a gene or nucleotide sequence modifiedthrough the use of New Breeding Techniques.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date

Immunity to disease(s) and or insect(s). A Brassica napus L. var.pabularia (DC.) plant which is not subject to attack or infection byspecific disease(s) and or insect(s) is considered immune.

Intermediate resistance to disease(s) and or insect(s). A Brassica napusL. var. pabularia (DC.) plant that restricts the growth and developmentof specific disease(s) and or insect(s), but may exhibit a greater rangeof symptoms or damage compared to resistant plants. Intermediateresistant plants will usually show less severe symptoms or damage thansusceptible plant varieties when grown under similar environmentalconditions and/or specific disease(s) and or insect(s) pressure, but mayhave heavy damage under heavy pressure. Intermediate resistant Brassicanapus L. var. pabularia (DC.) plants are not immune to the disease(s)and or insect(s).

Maturity (Date). Maturity refers to the stage when plants are of fullsize or optimum weight, and in marketable form or shape to be ofcommercial or economic value. In leaf types they range from 50-75 daysfrom time of seeding, depending upon the season of the year. In otherbaby leaf type, they range from 30 to 45 days after planting. InBrassica napus L. var. pabularia (DC.) types, they range from 25 to 40days from time of seeding.

New Breeding Techniques: New breeding techniques are said of various newtechnologies developed and/or used to create new characteristics inplants through genetic variation, the aim being targeted mutagenesis,targeted introduction of new genes or gene silencing (RdDM). Examples ofsuch new breeding techniques are targeted sequence changes facilitatedthru the use of Zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2 andZFN-3, see U.S. Pat. No. 9,145,565, incorporated by reference in itsentirety), Oligonucleotide directed mutagenesis (ODM), Cisgenesis andintragenesis, RNA-dependent DNA methylation (RdDM, which does notnecessarily change nucleotide sequence but can change the biologicalactivity of the sequence), Grafting (on GM rootstock), Reverse breeding,Agro-infiltration (agro-infiltration “sensu stricto”, agro-inoculation,floral dip), Transcription Activator-Like Effector Nucleases (TALENs,see U.S. Pat. Nos. 8,586,363 and 9,181,535, incorporated by reference intheir entireties), the CRISPR/Cas system (see U.S. Pat. Nos. 8,697,359;8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308;8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641, which are allhereby incorporated by reference), engineered meganuclease re-engineeredhoming endonucleases, DNA guided genome editing (Gao et al., NatureBiotechnology (2016), doi: 10.1038/nbt.3547, incorporated by referencein its entirety), and Synthetic genomics. A complete description of eachof these techniques can be found in the report made by the JointResearch Center (JRC) Institute for Prospective Technological Studies ofthe European Commission in 2011 and titled “New plant breedingtechniques—State-of-the-art and prospects for commercial development”,which is incorporated by reference in its entirety.

Plant adaptability. A plant having good plant adaptability means a plantthat will perform well in different growing conditions and seasons.

Plant Cell. As used herein, the term “plant cell” includes plant cellswhether isolated, in tissue culture or incorporated in a plant or plantpart.

Plant Part. As used herein, the term “plant part” includes plant cells,plant protoplasts, plant cell tissue cultures from which Brassica napusL. var. pabularia (DC.) plants can be regenerated, plant calli, plantclumps and plant cells that are intact in plants or parts of plants,such as embryos, pollen, ovules, flowers, seeds, rootstock, scions,stems, roots, anthers, pistils, root tips, leaves, meristematic cells,axillary buds, hypocotyls cotyledons, ovaries, seed coat endosperm andthe like. In some embodiments, the plant part at least comprises atleast one cell of said plant. In some embodiments, the plant part isfurther defined as a pollen, a meristem, a cell, or an ovule.

Quantitative Trait Loci (QTL) Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Resistance to disease(s) and or insect(s). A Brassica napus L. var.pabularia (DC.) plant that restricts highly the growth and developmentof specific disease(s) and or insect(s) under normal disease(s) and orinsect(s) attack pressure when compared to susceptible plants. TheseBrassica napus L. var. pabularia (DC.) plants can exhibit some symptomsor damage under heavy disease(s) and or insect(s) pressure.

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Hort. Society Enterprise Ltd. RHS Garden; Wisley,Woking, Surrey GU236QB, UK.

Single gene converted (conversion). Single gene converted (conversion)plants refer to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a plant are recovered in additionto the single gene transferred into the plant via the backcrossingtechnique or via genetic engineering. A single gene converted plant canalso be referred to a plant obtained though mutagenesis or through theuse of some new breeding techniques, whereas the single gene convertedplant has essentially all of the desired morphological and physiologicalcharacteristics of the original variety in addition to the single geneor nucleotide sequence muted or engineered through the new breedingtechniques.

Susceptible to disease(s) and or insect(s). A Brassica napus L. var.pabularia (DC.) plant that is susceptible to disease(s) and or insect(s)is defined as a Brassica napus L. var. pabularia (DC.) plant that hasthe inability to restrict the growth and development of specificdisease(s) and or insect(s). Plants that are susceptible will showdamage when infected and are more likely to have heavy damage undermoderate levels of specific disease(s) and or insect(s).

Tolerance to abiotic stresses. A Brassica napus L. var. pabularia (DC.)plant that is tolerant to abiotic stresses has the ability to endureabiotic stress without serious consequences for growth, appearance andyield.

Uniformity. Uniformity, as used herein, describes the similarity betweenplants or plant characteristics which can be a described by qualitativeor quantitative measurements.

Variety. A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties ofplants).

Brassica napus L. var. pabularia (DC.) Plants

Brassica napus L. var. pabularia (DC.), also known as Siberian kale, isa vegetable which has been grown for many years various places worldwideand which is becoming increasingly popular in the USA with the recentdevelopment of the industrial culture of ready-to-eat fresh products, insalad mix, dressing and garnishing in gourmet cuisines. This new trendhas lead to the development of baby-leaf products such as the ones fromKX-1. Delicate leaves are very attractive and appetizing, excellent fordressing gourmet food. They are grown at high concentration andharvested at very young or “baby leaf” stage, typically 15 to 40 daysafter planting. The planting is often done on wider 80 to 84 inch bedsand often containing up to one million plants per acre.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm.

In Brassica napus L. var. pabularia (DC.), these important traits mayinclude heavier texture, improved uniformity, especially of the leaves,stem and hypocotyl, higher seed yield, improved color, resistance todiseases and insects, tolerance to drought and heat, better post-harvestshelf-life of the leaves, better standing ability in the field, andbetter agronomic quality.

In some embodiments, particularly desirable traits that may beincorporated by this invention are improved resistance to differentviral, fungal, and bacterial pathogens. Important diseases include butare not limited to fungi such as Bremia lactucae, Fusarium oxysporum,Sclerotinia minor or sclerotorum, Botrytis cinerea, Rhizictonia solani,Microdochium panattonianum, Verticiulium dahliae, Erysiphe chicocearumor Pithium tracheiphilum, virus, such as LMV (lettuce mosaic virus),TSWV (tomato potted wilt virus), “Big vein” (composed of LBW (lettucebig vein virus) and MILV (miratiori lettuce virus)), TBSV (tomato bushystunt virus), LNSV (lettuce necrotic stunt virus), TuMV (turnip mosaicvirus), CMV (cucumber mosaic virus) or BWYV (beet western yellowsvirus), bacteria such as Pseudomonas, Xanthomonas or Rhizomonas.Improved resistance to insect pests is another desirable trait that maybe incorporated into new Brassica napus L. var. pabularia (DC.) plantsdeveloped by this invention. Insect pests affecting the various speciesof Brassica napus L. var. pabularia (DC.) include Nasonovia ribisnigri,Myzus persicae, Macrosiphum euphorbia, Nematodes pratylenchus ormeloidogyne, leafminers: Liriomyza huidobrensis or Pemphigus busarius.

Brassica napus L. var. pabularia (DC.) Breeding

The goal of Brassica napus L. var. pabularia (DC.) breeding is todevelop new, unique and superior Brassica napus L. var. pabularia (DC.)cultivar and hybrids. The breeder initially selects and crosses two ormore parental lines, followed by repeated selfing and selection,producing many new genetic combinations. Another method used to developnew, unique and superior Brassica napus L. var. pabularia (DC.) cultivaroccurs when the breeder selects and crosses two or more parental linesfollowed by haploid induction and chromosome doubling that result in thedevelopment of dihaploid cultivars. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations and the same is true for the utilization of thedihaploid breeding method.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarsdeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting cultivars he develops, exceptpossibly in a very gross and general fashion. This unpredictabilityresults in the expenditure of large research monies to develop superiornew Brassica napus L. var. pabularia (DC.) cultivars.

The development of commercial Brassica napus L. var. pabularia (DC.)cultivar requires the development and selection of Brassica napus L.var. pabularia (DC.) plants, the crossing of these plants, and theevaluation of the crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes or through the dihaploidbreeding method followed by the selection of desired phenotypes. The newcultivars are evaluated to determine which have commercial potential.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

i Pedigree Selection

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents possessing favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). The dihaploid breedingmethod could also be used. Selection of the best individuals is usuallybegun in the F₂ population; then, beginning in the F₃, the bestindividuals in the best families are selected. Replicated testing offamilies, or hybrid combinations involving individuals of thesefamilies, often follows in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease of new cultivars. Similarly, the development of new cultivarsthrough the dihaploid system requires the selection of the cultivarsfollowed by two to five years of testing in replicated plots.

ii Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

When the term Brassica napus L. var. pabularia (DC.) cultivar is used inthe context of the present invention, this also includes any Brassicanapus L. var. pabularia (DC.) cultivar plant where one or more desiredtrait has been introduced through backcrossing methods, whether suchtrait is a naturally occurring one, a mutant or a gene or a nucleotidesequence modified by the use of New Breeding Techniques. Backcrossingmethods can be used with the present invention to improve or introduceone or more characteristic into the Brassica napus L. var. pabularia(DC.) cultivar of the present invention. The term “backcrossing” as usedherein refers to the repeated crossing of a hybrid progeny back to therecurrent parent, i.e., backcrossing one, two, three, four, five, six,seven, eight, nine, or more times to the recurrent parent. The parentalBrassica napus L. var. pabularia (DC.) cultivar plant which contributesthe gene or the genes for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental Brassica napus L. var. pabularia(DC.) cultivar to which the gene or genes from the nonrecurrent parentare transferred is known as the recurrent parent as it is used forseveral rounds in the backcrossing protocol.

In a typical backcross protocol, the original cultivar of interest(recurrent parent) is crossed to a second cultivar (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a Brassica napus L.var. pabularia (DC.) plant is obtained wherein all the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, generally determined at a 5%significance level when grown in the same environmental conditions, inaddition to the gene or genes transferred from the nonrecurrent parent.It has to be noted that some, one, two, three or more, self-pollinationand growing of population might be included between two successivebackcrosses. Indeed, an appropriate selection in the population producedby the self-pollination, i.e. selection for the desired trait andphysiological and morphological characteristics of the recurrent parentmight be equivalent to one, two or even three additional backcrosses ina continuous series without rigorous selection, saving then time, moneyand effort to the breeder. A non-limiting example of such a protocolwould be the following: a) the first generation F1 produced by the crossof the recurrent parent A by the donor parent B is backcrossed to parentA, b) selection is practiced for the plants having the desired trait ofparent B, c) selected plant are self-pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,five, six, seven, eight, nine, or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and the physiological and morphological characteristics of parent A.Step (c) may or may not be repeated and included between the backcrossesof step (d).

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred parental line in order to find it then in the hybridmade thereof. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass visual inspection, simplecrossing, but also follow up of the characteristic(s) throughgenetically associated markers and molecular assisted breeding tools.For example, selection of progeny containing the transferred trait isdone by direct selection, visual inspection for a trait associated witha dominant allele, while the selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic in corn, require selfing the progeny to determine whichplant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new parental inbred of a hybridlettuce plant according to the invention but that can be improved bybackcrossing techniques. These genes are generally inherited through thenucleus.

In 1981, the backcross method of breeding counted for 17% of the totalbreeding effort for inbred line development in the United States,accordingly to, Hallauer, A. R. et al. (1988) “Corn Breeding” Corn andCorn Improvement, No. 18, pp. 463-481.

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R. W. Allard's 1960 book,published by John Wiley & Sons, Inc, Principles of Plant Breeding). Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a parental line of a hybrid variety with exactly theadaptation, yielding ability and quality characteristics of therecurrent parent but superior to that parent in the particularcharacteristic(s) for which the improvement program was undertaken.Therefore, this method provides the plant breeder with a high degree ofgenetic control of his work.

The method is scientifically exact because the morphological andagricultural features of the improved variety could be described inadvance and because the same variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method”, 1930 Jour. Amer. Soc.Agron., 22: 289-244).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will bemodified only with regards to genes being transferred, which aremaintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from ‘Hope’ wheat to ‘Bart wheat’ and even pursuing thebackcrosses with the transfer of bunt resistance to create ‘Bart 38’,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in California Commonalfalfa to create ‘Caliverde’. This new ‘Caliverde’ variety producedthrough the backcross process is indistinguishable from CaliforniaCommon except for its resistance to the three named diseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,colour characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, ‘Calady’, has been produced by Jones andDavis. As dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e. grain size. ‘Lady Wright’, along grain variety was used as the donor parent and ‘Coloro’, a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety ‘Calady’ was produced.

iii Single-Seed Descent and Multiple Seed Procedures

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F2 to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F2 individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F2 plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or moreflower containing seed from each plant in a population and blend themtogether to form a bulk seed lot. Part of the bulked seed is used toplant the next generation and part is put in reserve. The procedure hasbeen referred to as modified single-seed descent or the bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster than removing one seed from each flower by handfor the single seed procedure. The multiple-seed procedure also makes itpossible to plant the same number of seeds of a population eachgeneration of inbreeding. Enough seeds are harvested to make up forthose plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son, pp. 115-161; N. W. Simmonds, 1979, Principles of CropImprovement, Longman Group Limited; W. R. Fehr, 1987, Principles of CropDevelopment, Macmillan Publishing Co.; N. F. Jensen, 1988, PlantBreeding Methodology, John Wiley & Sons).

iii Open-Pollinated Populations

The improvement of open-pollinated populations of such crops as rye,maize and sugar beets, herbage grasses, legumes such as alfalfa andclover, and tropical tree crops such as cacao, coconuts, oil palm andsome rubber, depends essentially upon changing gene-frequencies towardsfixation of favorable alleles while maintaining a high (but far frommaximal) degree of heterozygosity.

Uniformity in such populations is impossible and trueness-to-type in anopen-pollinated variety is a statistical feature of the population as awhole, not a characteristic of individual plants. Thus, theheterogeneity of open-pollinated populations contrasts with thehomogeneity (or virtually so) of inbred lines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes to flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement.

First, there is the situation in which a population is changed en masseby a chosen selection procedure. The outcome is an improved populationthat is indefinitely propagable by random-mating within itself inisolation.

Second, the synthetic variety attains the same end result as populationimprovement, but is not itself propagable as such; it has to bereconstructed from parental lines or clones. These plant breedingprocedures for improving open-pollinated populations are well known tothose skilled in the art and comprehensive reviews of breedingprocedures routinely used for improving cross-pollinated plants areprovided in numerous texts and articles, including: Allard, Principlesof Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds, Principlesof Crop Improvement, Longman Group Limited (1979); Hallauer and Miranda,Quantitative Genetics in Maize Breeding, Iowa State University Press(1981); and, Jensen, Plant Breeding Methodology, John Wiley & Sons, Inc.(1988).

A) Mass Selection

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued. In massselection, desirable individual plants are chosen, harvested, and theseed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

B) Synthetics

A synthetic variety is produced by crossing inter se a number ofgenotypes selected for good combining ability in all possible hybridcombinations, with subsequent maintenance of the variety by openpollination. Whether parents are (more or less inbred) seed-propagatedlines, as in some sugar beet and beans (Vicia) or clones, as in herbagegrasses, clovers and alfalfa, makes no difference in principle. Parentsare selected on general combining ability, sometimes by test crosses ortoperosses, more generally by polycrosses. Parental seed lines may bedeliberately inbred (e.g. by selfing or sib crossing). However, even ifthe parents are not deliberately inbred, selection within lines duringline maintenance will ensure that some inbreeding occurs. Clonal parentswill, of course, remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or more cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enters a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

iv. Hybrids

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugarbeet, sunflower andbroccoli. Hybrids can be formed in a number of different ways, includingby crossing two parents directly (single cross hybrids), by crossing asingle cross hybrid with another parent (three-way or triple crosshybrids), or by crossing two different hybrids (four-way or double crosshybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F1progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F1 hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor and uniformity exhibited byF1 hybrids is lost in the next generation (F2). Consequently, seed fromF2 hybrid varieties is not used for planting stock.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

v. Bulk Segregation Analysis (BSA)

BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, isa method described by Michelmore et al. (Michelmore et al., 1991,Identification of markers linked to disease-resistance genes by bulkedsegregant analysis: a rapid method to detect markers in specific genomicregions by using segregating populations. Proceedings of the NationalAcademy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie etal., 1999, Journal of Experimental Botany, 50(337): 1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F2, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to virus), and theother from the individuals having reversed phenotype (e.g., susceptibleto virus), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs, SNPs or SSRs). Markers linkedto the phenotype can be identified and used for breeding or QTL mapping.

vi. Hand-Pollination Method

Hand pollination describes the crossing of plants via the deliberatefertilization of female ovules with pollen from a desired male parentplant. In some embodiments the donor or recipient female parent and thedonor or recipient male parent line are planted in the same field. Insome embodiments the donor or recipient female parent and the donor orrecipient male parent line are planted in the same greenhouse. Theinbred male parent can be planted earlier than the female parent toensure adequate pollen supply at the pollination time. In someembodiments, the male parent and female parent can be planted at a ratioof 1 male parent to 4-10 female parents. Pollination is started when thefemale parent flower is ready to be fertilized. Female flower buds thatare ready to open in the following days are identified, covered withpaper cups or small paper bags that prevent bee or any other insect fromvisiting the female flowers, and marked with any kind of material thatcan be easily seen the next morning. The male flowers of the male parentare collected in the early morning before they are open and visited bypollinating insects. The covered female flowers of the female parent,which have opened, are un-covered and pollinated with the collectedfresh male flowers of the male parent, starting as soon as the maleflower sheds pollen. The pollinated female flowers are again coveredafter pollination to prevent bees and any other insects visit. Thepollinated female flowers are also marked. The marked flowers areharvested. In some embodiments, the male pollen used for fertilizationhas been previously collected and stored.

vii. Bee-Pollination Method

Using the bee-pollination method, the parent plants are usually plantedwithin close proximity. In some embodiments more female plants areplanted to allow for a greater production of seed. Insects are placed inthe field or greenhouses for transfer of pollen from the male parent tothe female flowers of the female parent.

viii. Targeting Induced Local Lesions in Genomes (TILLING)

Breeding schemes of the present application can include crosses withTILLING® plant cultivars. TILLING® is a method in molecular biology thatallows directed identification of mutations in a specific gene. TILLING®was introduced in 2000, using the model plant Arabidopsis thaliana.TILLING® has since been used as a reverse genetics method in otherorganisms such as zebrafish, corn, wheat, rice, soybean, tomato andlettuce.

The method combines a standard and efficient technique of mutagenesiswith a chemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with asensitive DNA screening-technique that identifies single base mutations(also called point mutations) in a target gene. EcoTILLING is a methodthat uses TILLING® techniques to look for natural mutations inindividuals, usually for population genetics analysis (see Comai, etal., 2003 The Plant Journal 37, 778-786; Gilchrist et al. 2006 Mol.Ecol. 15, 1367-1378; Mejlhede et al. 2006 Plant Breeding 125, 461-467;Nieto et al. 2007 BMC Plant Biology 7, 34-42, each of which isincorporated by reference hereby for all purposes). DEcoTILLING is amodification of TILLING® and EcoTILLING which uses an inexpensive methodto identify fragments (Garvin et al., 2007, DEco-TILLING: An inexpensivemethod for SNP discovery that reduces ascertainment bias. MolecularEcology Notes 7, 735-746).

The TILLING® method relies on the formation of heteroduplexes that areformed when multiple alleles (which could be from a heterozygote or apool of multiple homozygotes and heterozygotes) are amplified in a PCR,heated, and then slowly cooled. As DNA bases are not pairing at themismatch of the two DNA strands (the induced mutation in TILLING® or thenatural mutation or SNP in EcoTILLING), they provoke shape change in thedouble strand DNA fragment which is then cleaved by single strandednucleases. The products are then separated by size on several differentplatforms.

Several TILLING® centers exists over the world that focus onagriculturally important species: UC Davis (USA), focusing on Rice;Purdue University (USA), focusing on Maize; University of BritishColumbia (CA), focusing on Brassica napus L. var. pabularia (DC.); JohnInnes Centre (UK), focusing on Brassica rapa; Fred Hutchinson CancerResearch, focusing on Arabidopsis; Southern Illinois University (USA),focusing on Soybean; John Innes Centre (UK), focusing on Lotus andMedicago ; and INRA (France), focusing on Pea and Tomato.

More detailed description on methods and compositions on TILLING® can befound in U.S. Pat. No. 5,994,075, US 2004/0053236 A1, WO 2005/055704,and WO 2005/048692, each of which is hereby incorporated by referencefor all purposes.

Thus in some embodiments, the breeding methods of the present disclosureinclude breeding with one or more TILLING plant lines with one or moreidentified mutations.

viii Mutation Breeding

Mutation breeding is another method of introducing new variation andsubsequent traits into plants. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means or mutating agents including temperature,long-term seed storage, tissue culture conditions, radiation (such asX-rays, Gamma rays, neutrons, Beta radiation, or ultraviolet radiation),chemical mutagens (such as base analogs like 5-bromo-uracil),antibiotics, alkylating agents (such as sulfur mustards, nitrogenmustards, epoxides, ethyleneamines, sulfates, sulfonates, sulfones, orlactones), azide, hydroxylamine, nitrous acid or acridines. Once adesired trait is observed through mutagenesis the trait may then beincorporated into existing germplasm by traditional breeding techniques.Details of mutation breeding can be found in W. R. Fehr, 1993,Principles of Cultivar Development, Macmillan Publishing Co.

New breeding techniques such as the ones involving the uses of ZincFinger Nucleases or oligonucleotide directed mutagenesis shall also beused to generate genetic variability and introduce new traits intovarieties.

ix. Double Haploids and Chromosome Doubling

One way to obtain homozygous plants without the need to cross twoparental lines followed by a long selection of the segregating progeny,and/or multiple backcrossings is to produce haploids and then double thechromosomes to form doubled haploids. Haploid plants can occurspontaneously, or may be artificially induced via chemical treatments orby crossing plants with inducer lines (Seymour et al. 2012, PNAS vol109, pg 4227-4232; Zhang et al., 2008 Plant Cell Rep. Dec 27(12)1851-60). The production of haploid progeny can occur via a variety ofmechanisms which can affect the distribution of chromosomes duringgamete formation. The chromosome complements of haploids sometimesdouble spontaneously to produce homozygous doubled haploids (DHs).Mixoploids, which are plants which contain cells having differentploidies, can sometimes arise and may represent plants that areundergoing chromosome doubling so as to spontaneously produce doubledhaploid tissues, organs, shoots, floral parts or plants. Another commontechnique is to induce the formation of double haploid plants with achromosome doubling treatment such as colchicine (El-Hennawy et al.,2011 Vol 56, issue 2 pg 63-72; Doubled Haploid Production in Crop Plants2003 edited by Maluszynski ISBN 1-4020-1544-5). The production ofdoubled haploid plants yields highly uniform cultivars and is especiallydesirable as an alternative to sexual inbreeding of longer-generationcrops. By producing doubled haploid progeny, the number of possible genecombinations for inherited traits is more manageable. Thus, an efficientdoubled haploid technology can significantly reduce the time and thecost of inbred and cultivar development.

x. Protoplast Fusion

In another method for breeding plants, protoplast fusion can also beused for the transfer of trait-conferring genomic material from a donorplant to a recipient plant. Protoplast fusion is an induced orspontaneous union, such as a somatic hybridization, between two or moreprotoplasts (cells of which the cell walls are removed by enzymatictreatment) to produce a single bi- or multi-nucleate cell. The fusedcell that may even be obtained with plant species that cannot beinterbred in nature is tissue cultured into a hybrid plant exhibitingthe desirable combination of traits.

xi. Embryo Rescue

Alternatively, embryo rescue may be employed in the transfer ofresistance-conferring genomic material from a donor plant to a recipientplant. Embryo rescue can be used as a procedure to isolate embryo's fromcrosses wherein plants fail to produce viable seed. In this process, thefertilized ovary or immature seed of a plant is tissue cultured tocreate new plants (see Pierik, 1999, In vitro culture of higher plants,Springer, ISBN 079235267x, 9780792352679, which is incorporated hereinby reference in its entirety).

Breeding Evaluation

Each breeding program can include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for use as parents in new commercial cultivars; those stilldeficient in a few traits may be used as parents to produce newpopulations for further selection.

In one embodiment, the plants are selected on the basis of one or morephenotypic traits. Skilled persons will readily appreciate that suchtraits include any observable characteristic of the plant, including forexample growth rate, height, weight, color, taste, smell, changes in theproduction of one or more compounds by the plant (including for example,metabolites, proteins, drugs, carbohydrates, oils, and any othercompounds).

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

It should be appreciated that in certain embodiments, plants may beselected based on the absence, suppression or inhibition of a certainfeature or trait (such as an undesirable feature or trait) as opposed tothe presence of a certain feature or trait (such as a desirable featureor trait).

Selecting plants based on genotypic information is also envisaged (forexample, including the pattern of plant gene expression, genotype, orpresence of genetic markers). Where the presence of one or more geneticmarker is assessed, the one or more marker may already be known and/orassociated with a particular characteristic of a plant; for example, amarker or markers may be associated with an increased growth rate ormetabolite profile. This information could be used in combination withassessment based on other characteristics in a method of the disclosureto select for a combination of different plant characteristics that maybe desirable. Such techniques may be used to identify novel quantitativetrait loci (QTLs). By way of example, plants may be selected based ongrowth rate, size (including but not limited to weight, height, leafsize, stem size, branching pattern, or the size of any part of theplant), general health, survival, tolerance to adverse physicalenvironments and/or any other characteristic, as described hereinbefore.

Further non-limiting examples include selecting plants based on: speedof seed germination; quantity of biomass produced; increased root,and/or leaf/shoot growth that leads to an increased yield (herbage orgrain or fiber or oil, or fruit or leaves) or biomass production;effects on plant growth that results in an increased seed yield for acrop; effects on plant growth which result in an increased yield;effects on plant growth that lead to an increased resistance ortolerance to disease including fungal, viral or bacterial diseases, tomycoplasma or to pests such as insects, mites or nematodes in whichdamage is measured by decreased foliar symptoms such as the incidence ofbacterial or fungal lesions, or area of damaged foliage or reduction inthe numbers of nematode cysts or galls on plant roots, or improvementsin plant yield in the presence of such plant pests and diseases; effectson plant growth that lead to increased metabolite yields; effects onplant growth that lead to improved aesthetic appeal which may beparticularly important in plants grown for their form, color or taste,for example the color intensity of Brassica napus L. var. pabularia(DC.) leaves, or the taste of said leaves.

Molecular Breeding Evaluation Techniques

Selection of plants based on phenotypic or genotypic information may beperformed using techniques such as, but not limited to: high through-putscreening of chemical components of plant origin, sequencing techniquesincluding high through-put sequencing of genetic material, differentialdisplay techniques (including DDRT-PCR, and DD-PCR), nucleic acidmicroarray techniques, RNA-seq (transcriptome sequencing), qRTPCR(quantitative real time PCR).

In one embodiment, the evaluating step of a plant breeding programinvolves the identification of desirable traits in progeny plants.Progeny plants can be grown in, or exposed to conditions designed toemphasize a particular trait (e.g. drought conditions for droughttolerance, lower temperatures for freezing tolerant traits). Progenyplants with the highest scores for a particular trait may be used forsubsequent breeding steps.

In some embodiments, plants selected from the evaluation step canexhibit a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120% or more improvement in aparticular plant trait compared to a control plant.

In other embodiments, the evaluating step of plant breeding comprisesone or more molecular biological tests for genes or other markers. Forexample, the molecular biological test can involve probe hybridizationand/or amplification of nucleic acid (e.g., measuring nucleic aciddensity by Northern or Southern hybridization, PCR) and/or immunologicaldetection (e.g., measuring protein density, such as precipitation andagglutination tests, ELISA (e.g., Lateral Flow test or DAS-ELISA),Western blot, immune labeling, immunosorbent electron microscopy (ISEM),and/or dot blot).

The procedure to perform a nucleic acid hybridization, an amplificationof nucleic acid (e.g., PCR, RT-PCR) or an immunological detection (e.g.,precipitation and agglutination tests, ELISA (e.g., Lateral F1 ow testor DAS-ELISA), Western blot, RIA, immunogold or immunofluorescentlabeling, immunosorbent electron microscopy (ISEM), and/or dot blottests) are performed as described elsewhere herein and well-known by oneskilled in the art.

In one embodiment, the evaluating step comprises PCR (semi-quantitativeor quantitative), wherein primers are used to amplify one or morenucleic acid sequences of a desirable gene, or a nucleic acid associatedwith said gene or QTLor a desirable trait (e.g., a co-segregatingnucleic acid, or other marker).

In another embodiment, the evaluating step comprises immunologicaldetection (e.g., precipitation and agglutination tests, ELISA (e.g.,Lateral F1 ow test or DAS-ELISA), Western blot, RIA, immuno labeling(gold, fluorescent, or other detectable marker), immunosorbent electronmicroscopy (ISEM), and/or dot blot), wherein one or more gene ormarker-specific antibodies are used to detect one or more desirableproteins. In one embodiment, said specific antibody is selected from thegroup consisting of polyclonal antibodies, monoclonal antibodies,antibody fragments, and combination thereof.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be utilizedin the present disclosure to determine expression of a gene to assistduring the selection step of a breeding scheme. It is a variant ofpolymerase chain reaction (PCR), a laboratory technique commonly used inmolecular biology to generate many copies of a DNA sequence, a processtermed “amplification”. In RT-PCR, however, RNA strand is first reversetranscribed into its DNA complement (complementary DNA, or cDNA) usingthe enzyme reverse transcriptase, and the resulting cDNA is amplifiedusing traditional or real-time PCR.

RT-PCR utilizes a pair of primers, which are complementary to a definedsequence on each of the two strands of the mRNA. These primers are thenextended by a DNA polymerase and a copy of the strand is made after eachcycle, leading to logarithmic amplification.

RT-PCR includes three major steps. The first step is the reversetranscription (RT) where RNA is reverse transcribed to cDNA using areverse transcriptase and primers. This step is very important in orderto allow the performance of PCR since DNA polymerase can act only on DNAtemplates. The RT step can be performed either in the same tube with PCR(one-step PCR) or in a separate one (two-step PCR) using a temperaturebetween 40° C. and 50° C., depending on the properties of the reversetranscriptase used.

The next step involves the denaturation of the dsDNA at 95° C., so thatthe two strands separate and the primers can bind again at lowertemperatures and begin a new chain reaction. Then, the temperature isdecreased until it reaches the annealing temperature which can varydepending on the set of primers used, their concentration, the probe andits concentration (if used), and the cation concentration. The mainconsideration, of course, when choosing the optimal annealingtemperature is the melting temperature (Tm) of the primers and probes(if used). The annealing temperature chosen for a PCR depends directlyon length and composition of the primers. This is the result of thedifference of hydrogen bonds between A-T (2 bonds) and G-C (3 bonds). Anannealing temperature about 5 degrees below the lowest Tm of the pair ofprimers is usually used.

The final step of PCR amplification is the DNA extension from theprimers which is done by the thermostable Taq DNA polymerase usually at72° C., which is the optimal temperature for the polymerase to work. Thelength of the incubation at each temperature, the temperaturealterations and the number of cycles are controlled by a programmablethermal cycler. The analysis of the PCR products depends on the type ofPCR applied. If a conventional PCR is used, the PCR product is detectedusing for example agarose gel electrophoresis or other polymer gel likepolyacrylamide gels and ethidium bromide (or other nucleic acidstaining).

Conventional RT-PCR is a time-consuming technique with importantlimitations when compared to real time PCR techniques.. Furthermore, thespecificity of the assay is mainly determined by the primers, which cangive false-positive results. However, the most important issueconcerning conventional RT-PCR is the fact that it is a semi or even alow quantitative technique, where the amplicon can be visualized onlyafter the amplification ends.

Real time RT-PCR provides a method where the amplicons can be visualizedas the amplification progresses using a fluorescent reporter molecule.There are three major kinds of fluorescent reporters used in real timeRT-PCR, general nonspecific DNA Binding Dyes such as SYBR Green I,TaqMan Probes and Molecular Beacons (including Scorpions).

The real time PCR thermal cycler has a fluorescence detection threshold,below which it cannot discriminate the difference between amplificationgenerated signal and background noise. On the other hand, thefluorescence increases as the amplification progresses and theinstrument performs data acquisition during the annealing step of eachcycle. The number of amplicons will reach the detection baseline after aspecific cycle, which depends on the initial concentration of the targetDNA sequence. The cycle at which the instrument can discriminate theamplification generated fluorescence from the background noise is calledthe threshold cycle (Ct). The higher is the initial DNA concentration,the lower its Ct will be.

Other forms of nucleic acid detection can include next generationsequencing methods such as DNA SEQ or RNA SEQ using any known sequencingplatform including, but not limited to: Roche 454, Solexa GenomeAnalyzer, AB SOLiD, Illumina GA/HiSeq, Ion PGM, Mi Seq, among others(Liu et al,. 2012 Journal of Biomedicine and Biotechnology Volume 2012ID 251364; Franca et al., 2002 Quarterly Reviews of Biophysics 35 pg.169-200; Mardis 2008 Genomics and Human Genetics vol 9 pg 387-402).

In other embodiments, nucleic acids may be detected with other highthroughput hybridization technologies including microarrays, gene chips,LNA probes, nanoStrings, and fluorescence polarization detection amongothers.

In some embodiments, detection of markers can be achieved at an earlystage of plant growth by harvesting a small tissue sample (e.g., branch,or leaf disk). This approach is preferable when working with largepopulations as it allows breeders to weed out undesirable progeny at anearly stage and conserve growth space and resources for progeny whichshow more promise. In some embodiments the detection of markers isautomated, such that the detection and storage of marker data is handledby a machine. Recent advances in robotics have also led to full serviceanalysis tools capable of handling nucleic acid/protein markerextractions, detection, storage and analysis.

Quantitative Trait Loci

Breeding schemes of the present application can include crosses betweendonor and recipient plants. In some embodiments said donor plantscontain a gene or genes of interest which may confer the plant with adesirable phenotype. The recipient line can be an elite line or cultivarhaving certain favorite traits such for commercial production. In oneembodiment, the elite line may contain other genes that also impart saidline with the desired phenotype. When crossed together, the donor andrecipient plant may create a progeny plant with combined desirable lociwhich may provide quantitatively additive effect of a particularcharacteristic. In that case, QTL mapping can be involved to facilitatethe breeding process.

A QTL (quantitative trait locus) mapping can be applied to determine theparts of the donor plant's genome conferring the desirable phenotype,and facilitate the breeding methods. Inheritance of quantitative traitsor polygenic inheritance refers to the inheritance of a phenotypiccharacteristic that varies in degree and can be attributed to theinteractions between two or more genes and their environment. Though notnecessarily genes themselves, quantitative trait loci (QTLs) arestretches of DNA that are closely linked to the genes that underlie thetrait in question. QTLs can be molecularly identified to help mapregions of the genome that contain genes involved in specifying aquantitative trait. This can be an early step in identifying andsequencing these genes.

Typically, QTLs underlie continuous traits (those traits that varycontinuously, e.g. yield, height, level of resistance to virus, etc.) asopposed to discrete traits (traits that have two or several charactervalues, e.g. smooth vs. wrinkled peas used by Mendel in hisexperiments). Moreover, a single phenotypic trait is usually determinedby many genes. Consequently, many QTLs are associated with a singletrait.

A quantitative trait locus (QTL) is a region of DNA that is associatedwith a particular phenotypic trait . Knowing the number of QTLs thatexplains variation in the phenotypic trait tells about the geneticarchitecture of a trait. It may tell that a trait is controlled by manygenes of small effect, or by a few genes of large effect or by a severalgenes of small effect and few genes of larger effect.

Another use of QTLs is to identify candidate genes underlying a trait.Once a region of DNA is identified as contributing to a phenotype, itcan be sequenced. The DNA sequence of any genes in this region can thenbe compared to a database of DNA for genes whose function is alreadyknown.

In a recent development, classical QTL analyses are combined with geneexpression profiling i.e. by DNA microarrays. Such expression QTLs(e-QTLs) describes cis- and trans-controlling elements for theexpression of often disease-associated genes. Observed epistatic effectshave been found beneficial to identify the gene responsible by across-validation of genes within the interacting loci with metabolicpathway- and scientific literature databases.

QTL mapping is the statistical study of the alleles that occur in alocus and the phenotypes (physical forms or traits) that they produce(see, Meksem and Kahl, The handbook of plant genome mapping: genetic andphysical mapping, 2005, Wiley-VCH, ISBN 3527311165, 9783527311163).Because most traits of interest are governed by more than one gene,defining and studying the entire locus of genes related to a trait giveshope of understanding what effect the genotype of an individual mighthave in the real world.

Statistical analysis is required to demonstrate that different genesinteract with one another and to determine whether they produce asignificant effect on the phenotype. QTLs identify a particular regionof the genome as containing one or several genes, i.e. a cluster ofgenes that is associated with the trait being assayed or measured. Theyare shown as intervals across a chromosome, where the probability ofassociation is plotted for each marker used in the mapping experiment.

To begin, a set of genetic markers must be developed for the species inquestion. A marker is an identifiable region of variable DNA. Biologistsare interested in understanding the genetic basis of phenotypes(physical traits). The aim is to find a marker that is significantlymore likely to co-occur with the trait than expected by chance, that is,a marker that has a statistical association with the trait. Ideally,they would be able to find the specific gene or genes in question, butthis is a long and difficult undertaking. Instead, they can more readilyfind regions of DNA that are very close to the genes in question. When aQTL is found, it is often not the actual gene underlying the phenotypictrait, but rather a region of DNA that is closely linked with the gene.

For organisms whose genomes are known, one might now try to excludegenes in the identified region whose function is known with somecertainty not to be connected with the trait in question. If the genomeis not available, it may be an option to sequence the identified regionand determine the putative functions of genes by their similarity togenes with known function, usually in other genomes. This can be doneusing BLAST, an online tool that allows users to enter a primarysequence and search for similar sequences within the BLAST database ofgenes from various organisms.

Another interest of statistical geneticists using QTL mapping is todetermine the complexity of the genetic architecture underlying aphenotypic trait. For example, they may be interested in knowing whethera phenotype is shaped by many independent loci, or by a few loci, andhow do those loci interact. This can provide information on how thephenotype may be evolving.

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization is possible due to DNA-DNAhybridization techniques (RFLP) and/or due to techniques using thepolymerase chain reaction (e.g. STS, SNPs, microsatellites, AFLP). Alldifferences between two parental genotypes will segregate in a mappingpopulation based on the cross of these parental genotypes. Thesegregation of the different markers may be compared and recombinationfrequencies can be calculated. The recombination frequencies ofmolecular markers on different chromosomes are generally 50%. Betweenmolecular markers located on the same chromosome the recombinationfrequency depends on the distance between the markers. A lowrecombination frequency usually corresponds to a low distance betweenmarkers on a chromosome. Comparing all recombination frequencies willresult in the most logical order of the molecular markers on thechromosomes. This most logical order can be depicted in a linkage map(Paterson, 1996, Genome Mapping in Plants. R.G. Landes, Austin.). Agroup of adjacent or contiguous markers on the linkage map that isassociated to a reduced disease incidence and/or a reduced lesion growthrate pinpoints the position of a QTL.

The nucleic acid sequence of a QTL may be determined by methods known tothe skilled person. For instance, a nucleic acid sequence comprisingsaid QTL or a resistance-conferring part thereof may be isolated from adonor plant by fragmenting the genome of said plant and selecting thosefragments harboring one or more markers indicative of said QTL.Subsequently, or alternatively, the marker sequences (or parts thereof)indicative of said QTL may be used as (PCR) amplification primers, inorder to amplify a nucleic acid sequence comprising said QTL from agenomic nucleic acid sample or a genome fragment obtained from saidplant. The amplified sequence may then be purified in order to obtainthe isolated QTL. The nucleotide sequence of the QTL, and/or of anyadditional markers comprised therein, may then be obtained by standardsequencing methods.

One or more such QTLs associated with a desirable trait in a donor plantcan be transferred to a recipient plant to incorporate the desirabletrait into progeny plants by transferring and/or breeding methods.

In one embodiment, an advanced backcross QTL analysis (AB-QTL) is usedto discover the nucleotide sequence or the QTLs responsible for theresistance of a plant. Such method was proposed by Tanksley and Nelsonin 1996 (Tanksley and Nelson, 1996, Advanced backcross QTL analysis: amethod for simultaneous discovery and transfer of valuable QTL fromun-adapted germplasm into elite breeding lines. Theor Appl Genet92:191-203) as a new breeding method that integrates the process of QTLdiscovery with variety development, by simultaneously identifying andtransferring useful QTL alleles from un-adapted (e.g., land races, wildspecies) to elite germplasm, thus broadening the genetic diversityavailable for breeding. AB-QTL strategy was initially developed andtested in tomato, and has been adapted for use in other crops includingrice, maize, wheat, pepper, barley, and bean. Once favorable QTL allelesare detected, only a few additional marker-assisted generations arerequired to generate near isogenic lines (NILs) or introgression lines(ILs) that can be field tested in order to confirm the QTL effect andsubsequently used for variety development.

Isogenic lines in which favorable QTL alleles have been fixed can begenerated by systematic backcrossing and introgressing of marker-defineddonor segments in the recurrent parent background. These isogenic linesare referred to as near isogenic lines (NILs), introgression lines(ILs), backcross inbred lines (BILs), backcross recombinant inbred lines(BCRIL), recombinant chromosome substitution lines (RCSLs), chromosomesegment substitution lines (CSSLs), and stepped aligned inbredrecombinant strains (STAIRSs). An introgression line in plant molecularbiology is a line of a crop species that contains genetic materialderived from a similar species. ILs represent NILs with relatively largeaverage introgression length, while BILs and BCRILs are backcrosspopulations generally containing multiple donor introgressions per line.As used herein, the term “introgression lines or ILs” refers to plantlines containing a single marker defined homozygous donor segment, andthe term “pre-ILs” refers to lines which still contain multiplehomozygous and/or heterozygous donor segments.

To enhance the rate of progress of introgression breeding, a geneticinfrastructure of exotic libraries can be developed. Such an exoticlibrary comprises a set of introgression lines, each of which has asingle, possibly homozygous, marker-defined chromosomal segment thatoriginates from a donor exotic parent, in an otherwise homogenous elitegenetic background, so that the entire donor genome would be representedin a set of introgression lines. A collection of such introgressionlines is referred as libraries of introgression lines or IL libraries(ILLs). The lines of an ILL cover usually the complete genome of thedonor, or the part of interest. Introgression lines allow the study ofquantitative trait loci, but also the creation of new varieties byintroducing exotic traits. High resolution mapping of QTL using ILLsenable breeders to assess whether the effect on the phenotype is due toa single QTL or to several tightly linked QTL affecting the same trait.In addition, sub-ILs can be developed to discover molecular markerswhich are more tightly linked to the QTL of interest, which can be usedfor marker-assisted breeding (MAB). Multiple introgression lines can bedeveloped when the introgression of a single QTL is not sufficient toresult in a substantial improvement in agriculturally important traits(Gur and Zamir, Unused natural variation can lift yield barriers inplant breeding, 2004, PLoS Biol.;2(10):e245).

Tissue Culture

As it is well known in the art, tissue culture of Brassica napus L. var.pabularia (DC.) can be used for the in vitro regeneration of Brassicanapus L. var. pabularia (DC.) plants. Tissues cultures of varioustissues of Brassica napus L. var. pabularia (DC.) and regeneration ofplants therefrom are well known. Thus, another aspect of this inventionis to provide cells which upon growth and differentiation produceBrassica napus L. var. pabularia (DC.) plants having the physiologicaland morphological characteristics of Brassica napus L. var. pabularia(DC.) cultivar KX-1.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, leaves, stems,roots, root tips, anthers, pistils, meristematic cells, axillary buds,ovaries, seed coat, endosperm, hypocotyls, cotyledons and the like.Means for preparing and maintaining plant tissue culture are well knownin the art. By way of example, a tissue culture comprising organs hasbeen used to produce regenerated plants. U.S. Patent Nos. 5,959,185,5,973,234, and 5,977,445 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

EXAMPLES

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

Example 1 Development of Brassica napus L. var. pabularia (DC.) cultivarKX-1

The Brassica napus L. var. pabularia (DC.) cultivar KX-1 is a darkpurple Brassica napus L. var. pabularia (DC.) variety obtained afterseveral rounds of crossing, self-pollination and selection.

Breeding History:

KX-1 was developed from a very unusual cross. KX-1 was previouslythought to be a Brassica oleracea variety, but was later determined tobe a Brassica napus variety through cytometry (e.g., it has the same DNAcontent as a Brassica napus plant).

The female parent is a proprietary plant of Applicant. The female plantis itself the result of the cross between a Brassica napus L. var.pabularia (DC.), (also generically known as Siberian Kale) with aBrassica oleracea var. acephala kale.

The male parent is a proprietary plant of Applicant. The male parent isitself the progeny of several complex crosses involving Brassica napusL. var. pabularia (DC.) and Brassica oleracea var alboglabra (alsogenerically known as Kailan). This male plant was itself ratherunexpected to obtain because of the incompatible blooming of the plants,as Siberian kale requires vernalization while kailan doesn't. Further,even after such crosses have been found possible by the presentinventor, only very few seeds were produced, due to geneticincompatibility. The cross, between the female and male plants, thatwill lead to KX-1 took place in Gilroy, Calif. on Spring of the firstyear.

The resulting seed was grown in fall of the first year at Gilroy. Twoselections were made and allowed to self, and the seed of each plant wascollected individually. The two batches of seed were assigned the sourcecodes B-1039-6-1 and, -2. The resulting seed was grown in fall of thesecond year at Gilroy. Nine selections were made from B-1039-6-2 ofwhich three survived to produce seed. The seed were assigned the sourcecode B-1158-23-1, -2 and -8. The resulting seed was grown in fall ofyear three at Gilroy. one selection was made and allowed to self, andthe seed of the plant was collected and assigned the source codeB-1242-119-1. The resulting seed was grown in fall of year four atGilroy. One selection was made from the resulting plants and the seed ofthe plant was collected and assigned the source code B-1392-129-1. Theresulting seed was grown in fall of year five at Gilroy. 127 plants wereselected, their seeds collected in bulk and those seeds became KX-1.

Some of the criteria used to select the Brassica napus L. var. pabularia(DC.) cultivar KX-1 in various generations include: uniformity, color ofthe leaves, texture of the leaves, leaf thickness, shape, smoothness andseed production, length of hypocotyl and stem. It is worth to note that;to the best knowledge of the inventor, KX-1 is the first Siberian Kalehaving a Dark Purple color of the leaves.

KX-1 Brassica napus L. var. pabularia (DC.) cultivar is similar to theRed Russian variety sold for example by Johnny's Selected Seeds, but hasnumerous differences: KX-1 is purple with a green petiole while RedRussian is green with a slightly pink petiole, the texture of KX-1 isheavier and more succulent than Red Russian, KX-1 is more uniform. Theleaf shape of KX-1 is similar to that of Red Russian but is more entire,with a greater leaf surface. Also, KX-1 produces seeds more readily andwith less vernalization than Red Russian.

The Brassica napus L. var. pabularia (DC.) cultivar KX-1 has shownuniformity and stability for the traits, within the limits ofenvironmental influence for the traits as described in the followingVariety Descriptive Information. No variant traits have been observed orare expected for agronomically important traits in Brassica napus L.var. pabularia (DC.) cultivar KX-1.

Brassica napus L. var. pabularia (DC.) cultivar KX-1 has the followingmorphologic and other characteristics, (based primarily on datacollected in California, all experiments done under the directsupervision of the applicant).

TABLE 1 Variety Description Information Plant: Brassica napus L. var.pabularia (DC.) Seed: Color: Black Cotyledon to Fourth Leaf Stage: Shapeof cotyledon: Reniform Shape of fourth leaf: Ovate Apical Margin: Deeplypinnipartite Basal Margin: Deeply pinnipartite Undulation: Slight GreenColor: Dark Purple Anthocyanin Distribution: Leaf blade AnthocyaninConcentration: Heavy Rolling (curvature parallel to the spine of theNone leaf): Cupping: None Reflexing (curvature perpendicular to thespine Slight of the leaf): Harvest-Mature Out Leaf, Head, Core: MarginIncision Depth: Medium Margin Indentation: Deeply pinnipartiteUndulation of the Apical Margin: Slight Green Color: Dark purpleAnthocyanin Distribution: Leaf blade Anthocyanin Concentration: HeavyGlossiness: Moderate Blistering: Slight Leaf Thickness: Medium ThickTrichomes: Nearly Absent (Smooth) Maturity (No. of Days of First Waterdate to NA Harvest): Outer Leaf Length (cm): full size harvest 20.5 OutLeaf Width (cm): full size harvest  6.5 Outer Leaf Length (cm): babyleaf size harvest NA Out Leaf Width (cm): baby leaf size harvest NADeposit Information

A deposit of the Brassica napus L. var. pabularia (DC.) seed of thisinvention is maintained by Shamrock Seed Company Inc., 3 Harris Place,Salinas, Calif. 93901-4593, USA. In addition, a sample of the Brassicanapus L. var. pabularia (DC.) seed of this invention has been depositedwith the National Collections of Industrial, Food and Marine Bacteria(NCIMB), 23 St Machar Drive, Aberdeen, Scotland, AB24 3RY, UnitedKingdom.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain of the present invention meetsthe criteria set forth in 37 C.F.R. 1.801-1.809, Applicants hereby makethe following statements regarding the deposited Brassica napus L. var.pabularia (DC.) cultivar KX-1 (deposited as NCIMB Accession No. 42703):

-   1. During the pendency of this application, access to the invention    will be afforded to the Commissioner upon request;-   2. All restrictions on availability to the public will be    irrevocably removed upon granting of the patent under conditions    specified in 37 CFR 1.808;-   3. The deposit will be maintained in a public repository for a    period of 30 years or 5 years after the last request or for the    effective life of the patent, whichever is longer;-   4. A test of the viability of the biological material at the time of    deposit will be conducted by the public depository under 37 C.F.R.    1.807; and-   5. The deposit will be replaced if it should ever become    unavailable. Access to this deposit will be available during the    pendency of this application to persons determined by the    Commissioner of Patents and Trademarks to be entitled thereto under    37 C.F.R. § 1.14 and 35 U.S.C. § 122. Upon allowance of any claims    in this application, all restrictions on the availability to the    public of the variety will be irrevocably removed by affording    access to a deposit of at least 2,500 seeds of the same variety with    the NCIMB.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

What is claimed is:
 1. A seed of Brassica napus L. var. pabularia (DC.) designated KX-1, wherein a representative sample of seed of said Brassica napus L. var. pabularia (DC.) having been deposited under NCIMB No
 42703. 2. A Brassica napus L. var. pabularia (DC.) plant, or a part thereof, or a plant cell thereof produced by growing the seed of claim
 1. 3. The Brassica napus L. var. pabularia (DC.) part of claim 2, wherein the Brassica napus L. var. pabularia (DC.) part is selected from the group consisting of: a leaf, a flower, an ovule, a cell and pollen.
 4. A Brassica napus L. var. pabularia (DC.) plant having all of the characteristics of Brassica napus L. var. pabularia (DC.) KX-1 listed in Table 1 when grown under the same environmental conditions, or a plant part or a plant cell thereof.
 5. A Brassica napus L. var. pabularia (DC.) plant, or a plant part thereof, having all of the physiological and morphological characteristics of Brassica napus L. var. pabularia (DC.) KX-1, wherein a representative sample of seed of said Brassica napus L. var. pabularia (DC.) having been deposited under NCIMB No
 42703. 6. A tissue culture of regenerable cells produced from the plant or plant part of claim 2, wherein a plant regenerated from the tissue culture has all of the characteristics of Brassica napus L. var. pabularia (DC.) KX-1 listed in Table 1 when grown under the same environmental conditions.
 7. A Brassica napus L. var. pabularia (DC.) plant regenerated from the tissue culture of claim 6, said plant having all the characteristics of Brassica napus L. var. pabularia (DC.) KX-1, wherein a representative sample of seed of said Brassica napus L. var. pabularia (DC.) having been deposited under NCIMB No
 42703. 8. A Brassica napus L. var. pabularia (DC.) leaf produced from the plant of claim
 2. 9. A method for producing a Brassica napus L. var. pabularia (DC.) leaf comprising a) growing the Brassica napus L. var. pabularia (DC.) plant of claim 2 to produce a Brassica napus L. var. pabularia (DC.) leaf, and b) harvesting said Brassica napus L. var. pabularia (DC.) leaf.
 10. A Brassica napus L. var. pabularia (DC.) leaf produced by the method of claim
 9. 11. A method for producing a Brassica seed comprising crossing the Brassica napus L. var. pabularia (DC.) plant of claim 2 with itself or a second, distinct Brassica plant.
 12. An F1 Brassica seed produced by the method of claim
 11. 13. A method for producing a Brassica napus L. var. pabularia (DC.) seed comprising self-pollinating the Brassica napus L. var. pabularia (DC.) plant of claim 2 and harvesting the resultant Brassica napus L. var. pabularia (DC.) seed.
 14. A Brassica napus L. var. pabularia (DC.) seed produced by the method of claim
 13. 15. A method of producing a Brassica plant derived from the Brassica napus L. var. pabularia (DC.) KX-1, the method comprising (a) crossing the plant of claim 2 with a second Brassica plant to produce a progeny plant.
 16. The method of claim 15 further comprising the step of: (b) crossing the progeny plant derived from Brassica napus L. var. pabularia (DC.) KX-1 with itself or a second plant to produce a seed of progeny plant of subsequent generation; (c) growing the progeny plant of the subsequent generation from the seed (d) crossing the progeny plant of the subsequent generation with itself or a second plant, to produce a Brassica plant derived from the Brassica napus L. var. pabularia (DC.) KX-1.
 17. The method of claim 16 further comprising the step of: (e) repeating steps (b) and/or (c) to produce a Brassica (DC.) plant derived from the Brassica napus L. var. pabularia (DC.) KX-1.
 18. The plant of claim 2 comprising a single locus conversion and otherwise essentially all the characteristics of KX-1 listed in Table 1 when grown in the same environmental conditions.
 19. The plant of claim 18 wherein the single locus conversion confers said plant with herbicide resistance.
 20. The plant of claim 18 wherein the single locus conversion is an artificially mutated gene or nucleotide sequence.
 21. The plant of claim 18 wherein the single locus conversion is a gene that has been modified through the use of new breeding techniques.
 22. A method of introducing a desired trait into Brassica napus L. var. pabularia (DC.) KX-1 comprising: (a) crossing a Brassica napus L. var. pabularia (DC.) KX-1 plant grown from Brassica napus L. var. pabularia (DC.) KX-1 seed, wherein a representative sample of seed has been deposited under NCIMB No. 42703, with another Brassica plant that comprises a desired trait to produce F1 progeny plants.
 23. The method of claim 22, further comprising: (b) selecting one or more progeny plants that have the desired trait to produce selected progeny plants; (c) crossing the selected progeny plants with the Brassica napus L. var. pabularia (DC.) KX-1 plants to produce backcross progeny plants; (d) selecting for backcross progeny plants that have the desired trait and all of the physiological and morphological characteristics of Brassica napus L. var. pabularia (DC.) KX-1 listed in Table 1 when grown in the same environmental conditions to produce selected backcross progeny plants; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny plants that comprise the desired trait and all of the physiological and morphological characteristics of Brassica napus L. var. pabularia (DC.) KX- 1 listed in Table 1 when grown in the same environmental conditions. 